The present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above. The present invention further concerns methods for screening for candidate activator or deactivators utilizing said amino acid sequences.
Prostate-specific antigen (PSA) is the most important tumor marker for early detection, staging, and monitoring of men with prostate cancer today. PSA testing has appreciable false-positive and false-negative results, particularly in the 2.5-10 ng/ml range. Measurement of the percentage of non-protein-bound (i.e. free) PSA in serum, which is lower in patients with prostate cancer, have been evaluated as a method for increasing the accuracy of PSA testing.
Thus measurement of PSA in serum, has been postulated as having potential clinical utility for increasing the sensitivity and specificity of PSA testing. Cutoff figures are affected by total PSA levels at prostate value. The prevalence rate of cancer in the screened population, depending on age, race, previous biopsy history etc., also influences the screening cutoffs. It has also been postulated that the percentage of free PSA may also correlate with a potential aggressiveness of early-stage prostate cancer. Thus, the level of free PSA may not only be used in order to diagnose prostate cancer, but also to predict the course of development of this cancer, and the patient""s prognosis, and decide on a suitable treatment regime.
Human kallikrein-2 gene (termed herein after: xe2x80x9cKLKxe2x80x9d which is also known as KLK-2) is transcribed from the same locus as the PSA and is also known to be prostate specific. It has been speculated that both PSA and KLK have common expression control such as common enhancer and/or promoter and both function as serine proteases.
In the following description and claims use will be made, at times, with a variety of terms, and the meaning of such terms as they should be construed in accordance with the invention is as follows:
xe2x80x9cProstate specific antigen (PSA) variantxe2x80x9dxe2x80x94the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, sequences having at least 70% identity to said sequence and fragments of the above sequences of least 20 b.p. long. SEQ ID NO: 1 to ID NO:5 are nucleic acid sequences which resulted from alternative splicing of the native and known PSA sequence appearing in HSPSAR and HUMPSANTIG (GenBank Acc. X05332 and M24543, respectively). It should be emphasized that the PSA variants of the invention are naturally occurring sequences resulting from the alternative splicing of the RNA transcribed from the PSA gene and not merely truncated or mutated forms of the gene. SEQ ID NO: 6 is an alternative splice variant of the human kallikrein-2 gene (KLK-2) appearing in GenBank as KLK2 under Accession Number NMxe2x80x94005551.
SEQ ID NO: 1xe2x80x94(PSALxe2x80x940): The nucleic acid sequence starting in position 4364 of the HUMPSANTIG up to position 7305, then a different sequence. The coded peptide (SEQ ID NO:7) starting identically to the original PSA for 16 aa, then a different sequence which is trancribed from the PSA intron between exons 1 and 2.
SEQ ID NO:2xe2x80x94(PSALxe2x80x941): Nucleic acid sequence identical to SEQ 1.
Peptide (SEQ ID NO: 8)xe2x80x94Starting in a Methionine 114 aa upstream from the original PSA, and has the same 16 aa identity and 3xe2x80x2 end as PSALxe2x80x940.
SEQ ID NO:3xe2x80x94(PSALxe2x80x942): Nucleic acid sequence which starts in same place as PSALxe2x80x940 but goes up to position 6336 of the HUMPSANTIG, then continues in a different sequence. Peptide (SEQ ID NO: 9)xe2x80x94Identical to PSALxe2x80x941 peptide.
SEQ ID NO: 4xe2x80x94(PSALxe2x80x945): Nucleic acid sequence which starts in same place as PSALxe2x80x940, goes up to position 6069 of HUMPSANTIG and end there (original intron). Peptide (SEQ ID NO:10)xe2x80x94Has same starting place as PSALxe2x80x941, the same 16 aa identity to PSA, then a different intron region translated.
SEQ ID NO: 5xe2x80x94(PSALxe2x80x946): Nucleic acid sequence starts in the same place as PSALxe2x80x940, goes up to position 5913 of HUMPSANTIG, then enter the original PSA exon # 2 and continues. Peptide (SEQ ID NO:11) has same starting place as PSALxe2x80x941, then enters the same identity region and continues as the original PSA until the end.
SEQ ID NO:6 is a splice variant of the KLK-2 that includes coding region from the original KLK-2 intron between exons 1 and 2. The term of xe2x80x9cPSA variantxe2x80x9d in the context of the present invention concerns splice variants of the known PSA gene as well as splice variants of the KLK-2 gene, which is also known to code for antigens specific to the prostate.
xe2x80x9cProstate specific antigen variant product (PSA variant product)xe2x80x94also referred at times as the xe2x80x9cPSA variant proteinxe2x80x9d or xe2x80x9cPSA variant polypeptidexe2x80x9dxe2x80x94an amino acid sequence coded by said PSA variant nucleic acid sequence. The amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein. An example of a PSA variant product is shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12, and includes also analogues of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments of this sequence having at least 10 amino acids. The products may be membrane associated or present in a free form in body fluids, for example in the serum.
xe2x80x9cNucleic acid sequencexe2x80x9dxe2x80x94a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
xe2x80x9cAmino acid sequencexe2x80x9dxe2x80x94a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
xe2x80x9cFragment of PSA variant productxe2x80x9d a sequence which is the same as part of but not all of the amino acid sequence of the PSA variant product.
xe2x80x9cFragments of PSA variant nucleic acid sequencexe2x80x9d a continuous portion, preferably of about 20 nucleic acid sequences of the PSA variant nucleic acid sequence (see below), which sequence does not appear in the original PSA.
xe2x80x9cConservative substitutionxe2x80x9dxe2x80x94refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.
xe2x80x9cNon-conservative substitutionxe2x80x9dxe2x80x94refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln.
xe2x80x9cChemically modifiedxe2x80x9dxe2x80x94when referring to the product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
xe2x80x9cBiologically activexe2x80x9dxe2x80x94refers to a PSA variant product which has the ability to serve as a marker of cancer, of predisposition to cancer, or of malignancy of a tumor.
xe2x80x9cImmunologically activexe2x80x9d defines the capability of a natural, recombinant or synthetic PSA variant product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. Thus, for example, a biologically active fragment of PSA variant product denotes a fragment which retains some or all of the biological properties of the PSA variant product, e.g the ability to serve as a marker for prostate cancer; an immunologically active fragment is a fragment which can bind specific anti-PSA variant product antibodies or xe2x80x9cdistinguishing antibodiesxe2x80x9d (see below) which can elicit an immune response which will generate such antibodies or cause proliferation of PSA variant product-specific immune cells. The fragment will also be denoted hereinafter as xe2x80x9cdistinguishing amino acid sequencexe2x80x9d.
xe2x80x9cOptimal alignmentxe2x80x9dxe2x80x94is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using a ktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from MacVector (TM), operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the xe2x80x9cgapxe2x80x9d of the first sequence).
xe2x80x9cHaving at least X% identityxe2x80x9dxe2x80x94with respect to two amino acid or nucleic acid sequence sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 90% amino acid sequence identity means that 90% of the amino acids in two or more optimally aligned polypeptide sequences are identical.
xe2x80x9cIsolated nucleic acid molecule having an PSA variant nucleic acid sequencexe2x80x9dxe2x80x94is a nucleic acid molecule that includes the coding PSA variant nucleic acid sequences. Said isolated nucleic acid molecule may include the PSA variant nucleic acid sequence as an independent insert; may include the PSA variant nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the PSA variant coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the PSA variant nucleic acid sequence may be in combination with non-coding sequences, e.g., introns or control elements, such as promoter and terminator elements or 5xe2x80x2 and/or 3xe2x80x2 untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the PSA variant protein coding sequence is a heterologous.
xe2x80x9cExpression vectorxe2x80x9dxe2x80x94refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
xe2x80x9cDeletionxe2x80x9dxe2x80x94is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
xe2x80x9cInsertionxe2x80x9d or xe2x80x9cadditionxe2x80x9dxe2x80x94is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
xe2x80x9cSubstitutionxe2x80x9dxe2x80x94replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non-conservative.
xe2x80x9cAntibodyxe2x80x9dxe2x80x94refers to IgG, IgM, IgD, IgA, and IgG antibody. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-PSA variant product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
xe2x80x9cDistinguishing antibodyxe2x80x9dxe2x80x94an antibody capable of binding only to the novel PSA variant product of the invention while not binding to the original PSA product, i.e. an antibody recognizing an additional amino acid sequence which appears only in the variant product of the invention and not in the original PSA sequence. This term may also refer at times to antibodies which binda sequence present in the original PSA and not present in the PSA variant product.
xe2x80x9cDistinguishing amino acid sequencexe2x80x9dxe2x80x94an amino acid sequence of at least two amino acids which are present only in the PSA variant of the invention and not in the original PSA of which are used to prepare the above distinguishing antibodies.
xe2x80x9cActivatorxe2x80x9dxe2x80x94as used herein, refers to a molecule which mimics the effect of the natural PSA variant product or at times even increases or prolongs the duration of the biological activity of said product, as compared to that induced by the natural product. The mechanism may be by binding to the PSA variant receptor, by prolonging the lifetime of the PSA variant, by increasing the activity of the PSA variant on its target, by increasing the affinity of PSA variant to its receptor, etc. Activators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which can bind to and activate the PSA variant product.
xe2x80x9cDeactivatorxe2x80x9dxe2x80x94refers to a molecule which modulates the activity of the PSA variant product in an opposite manner to that of the activator, by decreasing or shortening the duration of the biological activity of the PSA variant product. This may be done by blocking the binding of the PSA variant to its receptor, competitive or non competitive inhibitor, by causing rapid degradation of the PSA variant, etc. Deactivators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of said product.
xe2x80x9cTreating a diseasexe2x80x9dxe2x80x94refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring. In the context of the invention the disease is typically cancer and in particular prostate cancer.
xe2x80x9cDetectionxe2x80x9dxe2x80x94refers to a method of detection of a disease, such as prostate cancer. May be detection of an active disease or detection of a predisposition to a disease. By another alternative the detection may be capable of distinguishing between benign and malignant conditions. This term may also be used in connection with a method for evaluating the aggressiveness of a malignant state in order to correctly predict the prognosis of the patient, and in that case the detection may be used to assess the stage of the tumor.
xe2x80x9cProbexe2x80x9dxe2x80x94the PSA variant nucleic acid sequence, or a sequence (including fragments) complementary therewith, when used to detect presence of other similar sequences in a sample. The detection is carried out by identification of hybridization complexes between the probe and the assayed sequence. The probe may be attached to a solid support or to a detectable label. The probe may be a fragment of any one of the SEQ ID NO: 1 to SEQ ID NO: 6 (including a fragment of the non-coding region) which is of sufficient length to hybridize to the PSA variants at a level significantly different from the binding to the original PSA sequence. The probes may also be used to detect the polymorphisms described in the nucleic acid for the purpose of determining predisposition to cancer, especially prostate cancer in healthy individuals, and for detecting loss of heterozigosity in prostate tissues as part of a malignant transformation. The probes may be used in any method of performing this assay, including primer-specific PCR, allele-specific oligonucleotide assay, restriction fragment length differences, and mini-sequencing.
xe2x80x9cTargetingxe2x80x9dxe2x80x94directing a compound or drug to a desired cell population. Targeting is carried out by conjugating to the compound or drug an agent capable of binding specifically to the desired cell population, while not binding to non-desired cell populations. A specific example is targeting cytotoxic drugs directed only to tumor cells, more specifically directed to prostate tumor cells, for example, by conjugating the drug to an antibody of the invention.
xe2x80x9cOriginal PSA sequencexe2x80x9dxe2x80x94the known sequence of PSA as appears in GenBank HSPSAR locus and Acc # X05332, as well as to the known KLK-2 sequence as appears in GenBank KLK2 (NMxe2x80x94005551).
The present invention provides by its first aspect, a novel isolated nucleic acid molecule comprising or consisting of the coding sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 6, fragments of said coding sequence having at least 20 nucleic acids, or a molecule comprising a sequence having at 90% identity to any one of SEQ ID NO:1 to SEQ ID NO: 6. Preferably, the fragments should be such that they comprise sequences present in the PSA variants of the invention and not a sequence present in the original PSA (the term xe2x80x9coriginal PSAxe2x80x9d also includes the KLK-2 sequence).
These sequences are novel splice variants which results from alternative splicing of the original PSA sequence (this term according to the glossary refers also to the KLK-2 sequence).
The present invention further provides a protein or polypeptide comprising or consisting of an amino acid sequence encoded by any of the above nucleic acid sequences, termed herein xe2x80x9cPSA variant productxe2x80x9d, for example, an amino acid sequence having the sequence as depicted in any one of SEQ ID NO: 7 to SEQ ID NO: 12 fragments of the above amino acid sequence having a length of at least 10 amino acids, in particular fragments comprising sequences which do not appear in the original PSA sequence, as well as homologues of the amino acid sequences SEQ ID NO:7 to SEQ ID NO: 12 in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified.
The novel PSA variant products of the invention may have the same physiological activity as the original PSA peptide (this term refers also to the KLK-2 product) from which they are varied (although perhaps at a different level); may have an opposite physiological activity from the activity featured by the original peptide from which they are varied; may have a completely different, unrelated activity to the activity of the original from which they are varied; or alternatively may have no activity at all and this may lead to various diseases or pathological conditions.
The novel variants of the invention whether being nucleic acid or amino acid sequences may serve for detection purposes, i.e. their presence or level may be indicative of prostate cancer, predisposition to prostate cancer, malignancy of the cancer, stage of the cancer, or may be indicative to normal condition. Alternatively the ratio between the level of each variant and the level original PSA sequence from which it has been varied; the ratio of each variant to the or other variants; the total amount (sum) of two or more variants either by itself or compared to other variants; or the sum of two or more variants, may be indicative of cancer or predisposition to cancer in general, and prostate cancer or predisposition to prostate cancer in particular, as well as indicative of the malignancy of the cancer, its stage of development or of normal condition. The variants may be detected in blood or serum or in the prostate gland, the ovary, breast or salivary glands, which may share gene properties with the prostate gland. The variant products may be soluble or membrane bound.
For example, for detection purposes, it is possible to establish differential expression of the various variants in various tissues. A certain variant may be expressed mainly in one tissue, while the original PSA sequence may be expressed mainly in another tissue such as the prostate. Understanding of the distribution of the variants in various tissues may be helpful in basic research, for understanding the physiological function of the genes as well as may help in targeting pharmaceuticals or developing pharmaceuticals.
The study of the variants may also be helpful to distinguish various stages in the life cycles of the same type of cells which may also be helpful for development of pharmaceuticals for various pathological conditions in which cell cycles is un-normal, notably cancer. For example, various stages in the development of prostate cancer may be characterized by expression, or change in level of individual PSA variants of the invention.
Thus the detection may by determination of the presence or the level of expression of the variant within a specific cell population, comprising said presence or level between various cell types in a tissue, between different tissues and between individuals.
The present invention further provides nucleic acid molecule comprising or consisting of a sequence which encodes the above amino acid sequences, (including the fragments and analogs of the amino acid sequences). Due to the degenerative nature of the genetic code, a plurality of alternative nucleic acid sequences, beyond those of SEQ ID NO:1 to SEQ ID NO: 6, can code for the amino acid sequences of the invention. Those alternative nucleic acid sequences which code for the amino acid sequences codes by any one of the sequence SEQ ID NO: 1 to SEQ ID NO: 6 are also an aspect of the of the present invention.
The present invention further provides expression vectors and cloning vectors comprising any of the above nucleic acid sequences, as well as host cells transfected by said vectors.
The present invention still further provides pharmaceutical compositions comprising, as an active ingredient, said nucleic acid molecules, said expression vectors, or said protein or polypeptide.
These pharmaceutical compositions are suitable for the treatment of diseases and pathological conditions, which can be ameliorated or cured by raising the level of the PSA variant product, for example for the treatment of prostate cancer, or for inhibiting the transformation from prostate hyperplasia to malignancy. By another aspect, the present invention provides a nucleic acid molecule comprising or consisting of a non-coding sequence which is complementary to that of any one of SEQ ID NO:1 to SEQ ID NO: 6, or complementary to a sequence having at least 90% identity to said sequence or a fragment of said two sequences. The complementary sequence may be a DNA sequence which hybridizes with any one of the SEQ of ID NO:1 to SEQ ID NO: 6 or hybridizes to a portion of that sequence having a length sufficient to inhibit the transcription of the complementary sequence. The complementary sequence may be a DNA sequence which can be transcribed into an mRNA being an antisense to the mRNA transcribed from SEQ ID NO:1 to SEQ ID NO: 6 or into an mRNA which is an antisense to a fragment of the mRNA transcribed from SEQ ID NO:1 to SEQ ID NO: 6 which has a length sufficient to hybridize with the mRNA transcribed from SEQ ID NO: 1 to SEQ ID NO:6, so as to inhibit its translation. The complementary sequence may also be the mRNA or the fragment of the mRNA itself.
The nucleic acids of the invention may be used for therapeutic or diagnostic applications for example for detection of the expression of PSA variant in various tissues which may be indicative to the presence of prostate cancer, indicative of pre-disposition to prostate cancer, as well as indicative of the malignancy and hence the prognosis of the prostate cancer. The variants of the invention may also be indicative of other types of cancer from glands binding physiological similarity to the prostate gland such as ovary, breast, and salivary gland.
The present invention also provides expression vectors comprising any one of the above defined complementary nucleic acid sequences and host cells transfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention.
The invention also provides anti-PSA variant product antibodies, namely antibodies directed against the PSA variant product which specifically bind to said PSA variant product. Said antibodies are useful both for diagnostic and therapeutic purposes. For example said antibody may be used to detect the presence of prostate specific antigen-variant product in various tissues which may be indicative of the presence of prostate cancer of a predisposition for having prostate cancer, or of the malignancy of prostate cancer.
The present invention further concerns distinguishing antibodies which can bind only to a sequence present in the variants of the invention which is not present (as a continuous sequence) in the original PSA sequence. The present invention further concerns amino acid sequences for producing said distinguishing antibodies termed xe2x80x9cdistinguishing amino acid sequencesxe2x80x9d which are sequences present in the novel PSA variant and not present (as a continuous sequence) in the original PSA. An example of such a sequence is the sequence of positions 33-51 in SEQ ID NO:7 being:
Cys-Gln-Ala-Glu-Leu-Ser-Pro-Pro-Thr-Gln-His-Pro-Ser-Pro-Asp-Arg-Glu-Leu
The present invention also provides pharmaceutical compositions comprising, as an active ingredient, the nucleic acid molecules which comprise or consist of said complementary sequences, or of a vector comprising said complementary sequences. Alternatively, the pharmaceutical composition can comprise, as an active ingredient, said anti-PSA variant product antibodies, or said distinguishing antibodies.
The pharmaceutical compositions comprising said anti-PSA variant product antibodies, said distinguishing antibodies or the nucleic acid molecule comprising said complementary sequence, are suitable for the treatment of diseases and pathological conditions where a therapeutically beneficial effect may be achieved by neutralizing at least one of the PSA variants or decreasing the amount of the PSA variant product or blocking its binding to the receptor, for example, by the neutralizing effect of the antibodies, or by the effect of the antisense mRNA in decreasing expression level of the PSA variant product. An example of such a disease is prostate cancer. Furthermore, where the PSA variant is membrane bound, the anti-PSA variant antibodies may be used to target cytotoxic or cytostatic compounds to the tumor cells, in particular to prostate tumor cells. Since PSA variants may be produced specifically by prostate tumor cells, (and not normal prostate cells) and since this protein may be membrane associated, conjugates of anti-PSA variant antibodies and a drug can be targeted only to tumor cells and not harm healthy cells.
According to the third aspect of the invention the present invention provides methods for detecting the level of the transcript (mRNA) of said PSA variant product in a body fluid sample, or in a specific tissue sample, for example by use of probes comprising or consisting of said sequences (which may be a coding or uncoding sequence), as well as methods for detecting levels of expression of said product in tissue, e.g. by the use of antibodies capable of specifically reacting with the above amino acid sequences.
The method, according to this latter aspect, for detection of a nucleic acid sequence which encodes the PSA variant product in a biological sample, comprises the steps of:
(a) providing a probe comprising at least one of the nucleic acid sequence defined above;
(b) contacting the biological sample with said probe under conditions allowing hybridization of nucleic acid sequences thereby enabling formation of hybridization complexes;
(c) detecting hybridization complexes, wherein the presence of the complex indicates the presence of nucleic acid sequence encoding the PSA variant product in the biological sample.
The method as described above is qualitative, i.e. indicates whether the transcript is present in or absent from the sample. The method can also be quantitative, by determining the level of hybridization complexes and then calibrating said levels to determining levels of transcripts of the desired PSA variant in the sample.
Both qualitative and quantitative determination methods can be used for diagnostic, prognostic and therapy planning purposes.
By a preferred embodiment the probe is part of a nucleic acid chip used for detection purposes, i.e. the probe is a part of an array of probes each present in a known location on a solid support.
As indicated above the method may be utilized for detecting the presence of prostate cancer, detecting predisposition to prostate cancer, or evaluating the malignancy of prostate cancer, or assessing the development stage of the cancer.
The nucleic acid sequence used in the above method may be a DNA sequence, an RNA sequence, etc; it may be a coding or a sequence, or a non-coding sequence, or a sequence complementary thereto (for respective detection of RNA transcripts or coding-DNA sequences). By quantization of the level of hybridization complexes and calibrating the quantified results it is possible also to detect the level of the transcript in the sample.
The probes of the invention may be used to detect polymorphisms (in a specific individual or while screening a population) specifically for pre-disposition to cancer (especially prostate cancer) and loss-of-heterozigosity may be important for monitoring the development of the disease. Detection of disease predisposition or loss of heterozigosity in prostate tissue may be performed on either the coding or non-coding DNA sequence. One example of such a test is the determination of the exact sequence before position 5620 in GenBank HUMPSANTIG/1257 of SEQ ID 1 5620 (which is non coding and which contains an additional inserted A as compared to the native PSA), or testing a possible A to G substitution in position 5573 of HUMPSANTIG/1210 of SEQ ID 1. Both these sites may be indicative of cancer risk and useful in prognosis.
Methods for detecting mutations in the region coding for the PSA variant product are also provided, which may be methods carried-out in a binary fashion, namely merely detecting whether there is any mismatches between the normal PSA variant nucleic acid sequence and the one present in the sample, or carried-out by specifically detecting the nature and location of the mutation.
The present invention also concerns a method for detecting PSA variant product in a biological sample, comprising the steps of:
(a) contacting with said biological sample the antibody of the invention, thereby forming an antibody-antigen complex; and
(b) detecting said antibody-antigen complex wherein the presence of said antibody-antigen complex correlates with the presence of PSA variant product in said biological sample.
As indicated above, the method can be quantitized to determine the level or the amount of the PSA variant in the sample, alone or in comparison to the level of the original PSA amino acid sequence from which it was varied, and qualitative and quantitative results may be used for diagnostic, prognostic and therapy planning purposes.
By yet another aspect the invention also provides a method for identifying candidate compounds capable of binding to the PSA variant product and modulating its activity (being either activators or deactivators). The method includes:
(i) providing a protein or polypeptide comprising an amino acid sequence substantially as depicted in any one of SEQ ID NO: 7 to SEQ ID NO: 12, or a fragment of such a sequence;
(ii) contacting a candidate compound with said amino acid sequence;
(iii) measuring the physiological effect of said candidate compound on the activity of the amino acid sequences and selecting those compounds which show a significant effect on said physiological activity.
The activity of the amino acid which should be changed by the modulator (being either the activator or deactivator) may be for example the binding of the amino acid (PSA variant product) to its native, receptor. Any modulator which changes such an activity has an intersecting potential
The present invention also concerns compounds identified by the above methods described above, which compound may either be an activator of the serotonin-receptor like product or a deactivator thereof.